Clinical experience of the past decades has consistantly demonstrated that early tumor detection offers the best means of reducing high morbidity and mortality rates of cancer patients. Present clinical means of detecting neoplasms even with recent mdeical advances remains in many incidences a gross anatomical procedures relying upon various physical findings and radiographic diagnostic techniques to select a site for histologic sampling. Advances in radiography and the introduction of sonagraphy, xeroxgraphy and thermography have contributed significant improvement in detecting a large numbers of tumors. However, all these diagnostic means have inherent drawbacks and limitations, namely, a lack of sensitivity, specificity and reliability. Radiopharmaceuticals such as .sup.111 In-labeled bleomycin and .sup.67 Ga-gallium citrate are useful tumor imaging agents, but they lack specificity and sensitivity. Both agents are concentrated in inflammatory tissues and infectious lesions in addition to neoplasms.
Attempts to "mark" or "tag" cancer cells in order to differentiate them from normal tissue has been extensively investigated. Various fluorescent compounds such as tetracycline derivatives, acridine dyes and porphyrin compounds have been tried with mixed results. Of these, porphyrin compounds have shown remarkable affinity for neoplastic tissues.
Porphyrins and related analogs are complex tetrapyrrole compounds normally found in plants and in animals. They perform many vital biological functions by combining with metallic ions such as iron, magnesium, manganese, zinc, etc, . . . to form metalloporphyrins. Metalloporphyrins are essential for the normal metabolism of plant and animal. Many of these compounds exhibit strong fluorescence when exposed to an appropriate exciting light source.
Hematoporphyrin, an artifical porphyrin, is prepared by treating hemoglobin with concentrated sulfuric acid. It is a crude mixture of several porphyrins. Hematoporphyrin derivative(HPD), a recrystallized form of hematoporphyrin, is a complex mixture of hematoporphyrin diacetate, hematoporphyrin monoacetate, vinyl porphyrin, protoporphyrin, deuteroporphyrin and several addition analogs. The principle component in HPD is hematoporphyrin diacetate.(Lipson, R L, et al, J. Natl. Cancer Inst. 26: 1-11, 1961 and Clezy P. S., et al, Aust. J. Chem. 33: 585, 1980).
The preferential affinity of porphyrin compounds for various type of neoplasms has been known for more than four decades. When injected intravenously into tumor-bearing animal, a brilliant red-orange fluorescence is produced by ultra violet(UV) light activation of the porphyrin accumulated in the tumor. Hematoporphyrin derivative possesses higher tumor affinity than any other porphyrin compounds investigated. Successful clinical applications of HPD with human subjects in tumor detection have been documented in the literature (Sanderson, D R, et al, Cancer 30: 1368, 1972 and Kinsey, J H, et al. Mayo Clin. Proc. 53: 495, 1978).
Despite the initial optimism over possible diagnostic application of HPD in tumor detection, the usefulness of this agent is limited. This is primarily due to the fact that the HPD-fluorescence method involves invasive procedures. The fluorescence emitted by HPD must be activated in situ by a strong UV light source which requires highly sophisticated endoscopic fiberoptic equipments. Visual observation of the tissue fluorescence at best is subjective and varies widely from different investigators. Quenching of the fluorescence by normal tissue, body fluids and blood is a major obstacle in achieving significant reliability and reproducibility of this technique. Endoscopic procedures often produce tissue damages which lead to hemmorhage and subsequent masking of the tumor. Another major problem encountered is the inability to document photographically the fluorescence observed endoscopically. Complete reliance has to be placed on the visual interpretation and judgement of the endoscopist.
The use of radiolabeled HPD will eliminate most of the major problems encountered by the fluorescence-endoscopic procedures. Nuclear medicine techniques employing radiopharmaceuticals are simple and non-invasive. Following parenteral administration of the radiolabeled HPD, the radioactivity which concentrated in the tumor can be detected and documented on x-ray film by scintigraphic imaging means.
Several porphyrin compounds had been labeled with radionuclides such as .sup.64 Cu and .sup.57 Co. These radiolabeled porphyrins failed to achieve tumor localization in animal and human tumors (Wang T S T, et al, in Radiopharmaceuticals, Structure-Activity Relationship, Edited by R. P. Spencer, Grune and Stratton Press, New York 1981, pages 225-249). Earlier failures to localize neoplasms with radiolabeled porphyrins were attributed to: (1) poor labeling methodology; (2) alteration in biochemical property due to the labeling process; (3) a change in biochemical behavior of the parent porphyrin compound by the incorporation of the metallic ions; (4) in vivo instability of the labeled porphyrins; (5) the radionuclides used in the labeling process were incompatible with conventional scintigraphic imaging equipments.
At present, there are only two radionuclides of indium useful for medical applications. These are .sup.111 In and .sup.113m In. Of these, .sup.111 In possess the most ideal radioisotopic characteristics for scintigraphic imaging procedures. It is a pure gamma emitter with a physical half-life of 2.83 days. Its gamma energy of 173 keV and 247 keV photons are compatible with conventional imaging equipments. Because of its longer half-life, .sup.111 In-based radiopharmaceuticals are ideally suited for imaging studies that require observation period in days or weeks. Optimal delayed images can be obtained with a single injection of a small dose of the radiolabeled compound and yet produces the minimal amount of radiation health hazard to the patient.
Indium-113m is also a pure gamma emitter. It emits a monoenergetic gamma photon of 393 keV which is compatible with existing scintillation Anger cameras. It has a relatively short physical half-life of 1.65 hours. Indium-113m based radiopharmaceuticals are not suitable for imaging studies that require observation period of more than 6 hours.
The common method of labeling porphyrins with radionuclides involves the reflux reaction of a porphyrin with a radioactive metallic salt in an acidic or basic medium. Dilute hydrochloric acid(HCl), acetic acid or dilute base such as sodium hydroxide (NaOH) is used to dissolve the porphyrin and to act as the reaction medium. An aqueous solution of cobaltous chloride (.sup.57 CoCl.sub.2), cuprous chloride (.sup.64 CuCl.sub.2) or .sup.64 Cu-acetate is added to the porphyrin solution and reflux for 30 minutes to up to 24-48 hours depending on the reactivity of the porphyrin used in the labeling process. The pH of the radioactive admixture is then adjusted to 6-8 whenever possible without causing denaturation or precipitation of the radiolabeled porphyrin. In many incidences, the labeled product must remain in either acidic or basic condition in order to insure chemical and labeling stability.
Although the labeling process is quite simple, but the labeling yield is unsatisfactory, ranging from 10-40%. The final labeled product contains many radioactive impurities. These include free or unbound radionuclide, denatured by products and insoluble radiocolloids in the form of hydroxide such as .sup.57 Co(OH).sub.2 or .sup.64 Cu(OH).sub.2. Without extensive purification processes, these preparations are not useful or suitable for medical applications.
The present invention, that is, a chemical method of labeling porphyrins or hematoporphyrin derivative(HPD) with the radionuclides of indium, offers many advantages over earlier techniques. These include: (1) the labeling process can proceed in aqueous medium at neutral pH 7-8 condition without the problems of denaturation or decomposition; (2) the radionuclide is firmly bound to the porphyrin ligand; (3) the labeling yield is greater than 98% with consistant reproducibility and reliability; (4) the radiolabeled porphyrin is stable in vitro and in vivo as confirmed by radiochemical and anamal assays; (5) the labeled product is clear and freed from microcolloids contamination and can be given to patient by parenteral routes; (6) unlike other radiolabeled porphyrins, .sup.111 In- or .sup.113m In-labeled porphyrin is preferentially accumulated by neoplasms; (7) because of long half-life of .sup.111 In, the condition of the patient can be followed for days with a single injection of .sup.111 In-labeled porphyrin compound.